Cancer vaccines: Enhanced immunogenic modulation through therapeutic combinations

ABSTRACT

Therapeutic cancer vaccines have gained significant popularity in recent years as new approaches for specific oncologic indications emerge. Three therapeutic cancer vaccines are FDA approved and one is currently approved by the EMA as monotherapy with modest treatment effects. Combining therapeutic cancer vaccines with other treatment modalities like radiotherapy (RT), hormone therapy, immunotherapy, and/or chemotherapy have been investigated as a means to enhance immune response and treatment efficacy. There is growing preclinical and clinical data that combination of checkpoint inhibitors and vaccines can induce immunogenic intensification with favorable outcomes. Additionally, novel methods for identifying targetable neoantigens hold promise for personalized vaccine development. In this article, we review the rationale for various therapeutic combinations, clinical trial experiences, and future directions. We also highlight the most promising developments that could lead to approval of novel therapeutic cancer vaccines.

KEYWORDS:

• cancer vaccine
• combination immunotherapy
• immunogenic modulation
• immune-related response criteria
• immunotherapy

Introduction

While vaccines have historically been a preventative measure for infectious diseases and for prevention of virus-related cancers (i.e., hepatitis B virus [HBV] and human papilloma virus [HPV] vaccines), therapeutic cancer vaccines have generated significant interest within the medical and lay community as they offer the potential to direct a host's immune system against a tumor. There are three therapeutic cancer vaccines approved by the U.S. Food and Drug Administration (FDA): (1) Bacillus Calmettle-Guerin (TheraCys®) – a live attenuated strain of Mycobacterium bovis for non-muscle invasive bladder carcinoma; (2) Sipuleucel-T (Provenge®) – a dendritic cell (DC) vaccine for metastatic castration resistant prostate cancer (mCRPC); and (3) talimogene laherparepvec (T-VEC or Imlygic®) – an oncolytic viral-based vaccine for advanced melanoma. Approval was based on modest improvements in overall survival (sipuleucel-T and T-VEC), disease free survival (TheraCys), and a durable response rate (T-VEC).^1-3

In order to improve on these modest gains, tumor immune escape caused by natural selection of tumor cell clones lacking immunogenic antigens must be overcome.⁴ Successful tumor clones can persist via acquired defects, epigenetic silencing of various components involved in antigen processing, or by upregulating inhibitory receptors leading to exhaustion of effector T-cells.⁵

There are currently 369 open “cancer vaccine” studies on clinicaltrials.gov with 232 studies in the United States alone (as of 6/13/17). Numerous cancer vaccines have been tested in multiple solid as monotherapy or in combination with chemotherapy, radiation, or other immunotherapy agents. In this manuscript, we will first review experiences with combination approaches and then discuss strategies that we believe have the most promise.

Vaccine platforms

There are multiple therapeutic cancer vaccine platforms including peptide-based, protein-based, viral-based, recombinant vector including yeast-based and bacterial-based, whole tumor cell and pulsed dendritic cells(^6-12 Generally, most vaccines are well tolerated and have minimal side effects. Given the unique biology of different tumors types and the distinct variables that exist within an individual immune system, a discussion of optimal vaccine platform is beyond the scope of this review.

FDA approved cancer vaccines

The first FDA approved cancer vaccine was the intravesical BCG vaccine (TheraCys) in 1990 for the treatment and prophylaxis of primary or recurrent non-muscle invasive urothelial carcinoma following transuretheral resection.³ TheraCys prolonged disease-free survival (DFS) to 30 months in patients with bladder carcinoma in situ (CIS) and to 22.5 months in patients with Ta/T1 urothelial carcinoma, compared to 4.9 months DFS in CIS and 10.5 months in Ta/T1 patients treated with topical doxorubicin.^3,13

Sipuleucel-T (Provenge), an autologous DC vaccine containing a recombinant fusion protein, PA2024, that consists of prostate acid phosphatase (PAP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was FDA approved for patients with minimally symptomatic or asymptomatic mCRPC in 2010.¹ Sipuleucel-T has been shown to generate anti-tumor immune responses including PAP-specific T-cells and antigen cascade (immune response to antigens not contained in the vaccine).^14,15 A pivotal phase III study demonstrated a statistically significant 4.1 month improvement in median overall survival (OS); 25.8 months in the Sipuleucel-T group compared to 21.7 months in the placebo group.¹ Sipuleucel-T was initially approved by the European Medicines Association (EMA) in 2013 but was withdrawn in 2015 by the manufacturing company (Dendreon UK Ltd) who cited commercial reasons.

T-VEC (talimogene laherparepvec) is an oncolytic herpes virus in which two viral genes are deleted and that is modified to produce GM-CSF for enhancing immunogenicity. T-VEC was approved by the FDA and the EMA in 2015 for treatment of advanced melanoma based on data from the phase III OPTiM trial. The vaccine virus infects both cancer and normal cells but can only replicate within a cancer cell. Injected intralesionally, the vaccine is designed to produce a systemic anti-tumor effect.¹⁶ The OPTiM trial showed a higher durable response rate with T-VEC compared to GM-CSF alone (DRR; 16.3% vs 2.1%; p <0.001), as well as a higher overall response rate (ORR; 26.4% vs 5.7%, respectively) and a longer median OS (23.3 vs 18.9 months, p = 0.051) in patients with Stage IIIB, IIIC or IV M1a melanoma.²

The makings of an effective vaccine

Much has changed since 2009 when the NCI ranked 75 antigens thought to be important for an effective cancer vaccine antigen which included criteria such as good therapeutic function, ability to elicit T-cell and/or antibody responses and association with an oncogenic process.¹⁷ Broadly speaking, tumor antigens can be divided into tumor associated (TAA) or tumor specific (TSA) antigens. Since TAAs are expressed on both cancer and normal cells, it was thought their use would be hampered by generation of tolerance to high avidity TAA-specific T-cells.¹⁸ However, multiple studies have demonstrated that TAA-based vaccines can produce anti-tumor immune responses, albeit with only modest clinical benefit. Nonetheless, clinical experiences with PROSTVAC,^19,20 a PSA-targeted vaccine for prostate cancer, and Sipuleucel-T^14,15 which was discussed above, have shown that these TAA-based vaccines can generate an anti-tumor immune response and tumor-specific T-cells. A Phase III trial of PROSTVAC has completed accrual with results expected by the end of 2017 (NCT01322490).

Another promising TAA-based vaccine is NeuVax, which is desiged to prevent clinical recurrence in high risk, disease free patients with HER2+ breast cancer.^21,22 The NeuVax contains an immunogenic peptide called Nelipepimut-S (aka E75) from the HER2 protein and is combined with GM-CSF. Early phase trials have demonstrated clinical benefit in women with node-positive or high-risk node-negative HER2+ breast cancer.²² A Phase III study with NeuVax is currently ongoing (NCT01479244).

Use of TSA-targeted vaccines is attractive because such vaccines target antigens found only on tumor cells, and should theoretically limit issues with tolerance and toxicity. Identifying immunogenic tumor specific neoantigens is challenging and increasingly complicated since neoantigens are dynamic and change in response to various stimuli (i.e., treatment effects, immune infiltration, tumor mutations).^23,24 Furthermore, not every protein product of tumor-exclusive mutations will yield an immunogenic peptide antigen i.e. an epitope that will be presented on MHC and recognized by T-cells, making screening for T-cell epitopes labor intensive.

Yadav et al. reported a novel method for identifying immunogenic neoantigens.²⁵ Briefly, whole exome sequencing was performed on MC-38 and TRAMP-C1 murine tumor cell lines, followed by selection for high-confidence mutations based on overlap with RNA-Seq transcriptome analysis. Out of over 1,300 candidate amino acid mutations, 7 (all in MC-38) were confirmed by mass spectroscopy to be expressed on MHC class I. Manual verification with synthetic peptides narrowed to 6 epitopes. After peptide vaccination with adjuvant, tumor infiltrating lymphocytes (TILs) specific for the 3 out of 6 of the peptide's associated neoantigens were detected in MC-38 tumor-bearing mice. Compared to other TILs, they displayed an activated phenotype (PD-1 and TIM-3 high). In vivo, most mice vaccinated with the more immunogenic mutant peptides had no tumor growth following challenge with MC-38 inoculation, compared to controls. This approach provides an innovative pathway for developing personalized cancers vaccines, by selecting only target antigens from one's own tumor that are predicted to be immunogenic²⁶ and then incorporating into any of the various available vaccine platforms.

In addition to choosing a viable vaccine platform, selection of the appropriate patient population is also vital to effective therapeutic cancer vaccine design. In 2007 the Cancer Vaccine Consortium outlined recommendations for therapeutic vaccine trial design based on review of prior trials.^27,28 This group recommended using early stage disease and/or low volume disease. While many trials have evaluated therapeutic cancer vaccines in the advanced disease setting, it is worth noting that the only approved FDA therapeutic cancer vaccines are approved in the limited disease setting.^1-3

Vaccine combination strategies

Vaccination can induce antigen-specific T-cells;¹⁹ however, vaccines alone are seldom sufficient to induce a strong enough immune response for tumor eradication. The pharmaceutical pipeline continues to release a variety of investigational agents that modulate the immune response. Checkpoint inhibitors, immunoagonists and immunocytokines can induce a spectrum of alterations upon cancer and/or immune cells that can enhance immune destruction of tumor cells.^29-31 Additionally, there is evidence that combining therapeutic cancer vaccines with traditional modalities such as radiation, immunotherapy, hormone therapy and/or chemotherapy may be synergistic. (Table 2).

Table 1. Vaccine platforms (Table adapted from multiple sources^6-12).

Platform	Rationale	Immunogenicity	Toxicity	HLA Restriction	Pros	Cons
Peptide – Based	Elicit immunity to tumor associated self-antigens or tumor specific antigens	Low	Low	Yes	• Easy to produce with high purity in large amounts (e.g., cost effective)	• Only target one or a few epitopes – needs multiple peptides to prevent immune escape
					• High specificity through use of defined epitopes
						• Immunological compatibility only in patients with a specific HLA subtype.
					• No additional treatments/procedures for patients
					• Not pharmacologically active (little toxicity)	• Peptides with low affinity for MHC may be poorly immunogenic
					• Repeated booster vaccines to help with sustained immune response
						• Immune responses may be transient or of low magnitude
						• Often requires that it is given with immunoadjuvant to enhance immunogenicity
Protein – Based	Elicit immunity to tumor associated self-antigens or tumor specific antigens	Moderate	Low	No	• Multiple epitopes	• Give with immunoadjuvant to enhance immunogenicity
						• More costly than peptide-based vaccines
						• Higher antigen load
Viral – Based	Use natural ability to trigger immune responses and carry genetic material into cells for production of antigens	High	High	No	• Introduce target antigen into immune cells	• Immune response against priming virus requires different virus for a booster
					• Easy to produce on large scale
Bacteria – Based	Use natural ability to trigger immune responses and carry genetic material into cells for production of antigens	High	High	No	• Effective antigen delivery – express foreign antigens	• Risk of undesired infections (but may be treated with antibiotics)
					• Stimulate mucosal and systemic responses
					• Easy to produce on large scale
Dendritic Cells/ Antigen Presenting Cells	DC's are the body's most effective APCs Can have protein, peptide or viral infected cells	High	Low	Yes	• More control over APC stimulation and antigen presentation	• Logistically difficult (APC selection, ex vivo maturation)
					• HLA restricted but autologous cells	• Expensive – collection and manipulation
						• Need for a leukapheresis facility
						• Risk of leukapheresis (central line insertion, hypotension, electrolyte imbalances, vascular injury)
						• Difficult to ensure consistent vaccine production (varying leukapheresis yields, varying activated APCs, possible bacterial contamination)
Whole Tumor Cells	Deliver multiple relevant tumor antigens	Moderate	Low	No	• Broad array of antigens represented (minimizes immune escape)	• Cancer cells themselves are not that immunogenic
						• Autologous cell vaccines are labor-intensive and difficult to standardize
					• Readily produce allogenic cell lines on larger scale
					• Not HLA restricted due to whole proteins present

HLA Restriction – Different HLA subtypes present different antigens. Antigen is only immunological comparable in patients with a specific HLA subtype (i.e., HLA-A2+)

Table 2. Rationale for therapeutic cancer vaccine combinations.

	Rationale	References
Cytokines (IL, INF, TGF-β)	• Stimulate humoral and cellular immunity	32-37, 49, 50
	• Promote epithelial to mesenchymal transition
	• Promote differentiation of immature T-cells into Tregs and effectors T-cells
	• Promote dendritic cell maturation
	• Chemoattractant for neutrophils and MDSCs
Radiotherapy (External beam and radiopharmaceuticals)	• Enhance destruction of tumor cells via	29, 55-57, 61
	• upregulation of MHC, Fas, ICAM-1 and TAAs
	• Enhance vaccine-mediated tumor lysis
	• Increased inflammation and secretion of immunomodulatory cytokines
	• Sensitize tumor cells to immune-mediated killing
Checkpoint inhibitors (anti-CTLA-4/anti PD-1/PDL-1)	• Immunogenic intensification	77, 78, 93
	• Increased inflammation within the tumor
	• Reduction of Tumor burden
	• Activation of different T-cell population
Small molecules (TKIs/ HDACi)	• “Off-target” effects on immune cells (i.e., decreasing Tregs, decreasing MDSCs, increasing INF-g producing T-cells, and decreasing IL-4 producing T-cells	103-105, 108, 112
	• Sensitize cells to immune-mediated killing
	• Increase the protein expression of antigen presenting machinery
Endocrine Therapy	• Inducing thymic regeneration leading to increased production of naive T cells and CD4+ effector T cells.	115-118
	• Decrease Tregs within the tumor
Chemotherapy (including low dose CTX)	• Sensitize cells to immune-mediated killing	121-123,127,128,131,132,138,141
	• Decrease MDSCs and Tregs
	• Increase immune-supportive M1 macrophages, including CD4+ and CD8+ T-regs
	• Reduction of tumor burden
	• Induction of tumor immunsurveillance by NK cells
	• Enhance immunity by inhibiting Tregs, by enhancing DC maturation and by promoting of a durable T-cell memory response (low dose CTX)

Vaccine plus cytokines

Tumors often secrete their own immunosuppressive cytokines including TGF-ß, IL-4, IL-6 and IL-10. Co-administration of immunostimulatory cytokines with vaccines offers a potential means to augment the effect of vaccine derived effector T-cells. While data from large trials are still lacking, smaller clinical trials have investigated multiple cytokines in combination with vaccines, at varying schedules, in several malignancies.

Vaccines plus GM-CSF

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pro-inflammatory cytokine that stimulates humoral and cellular immunity.^32,33 Interestingly, two of the FDA-approved therapeutic cancer vaccines (Sipuleucel-T and T-VEC) are engineered to secrete GM-CSF. Hypothetically GM-CSF augments DC activity and thus enhances the immune response in each of these different platforms. However, its role, or lack thereof, in achieving the improved clinical outcomes seen with these agents is unclear. While GM-CSF is important for maturation of DC, activation and proliferation of T-cells, it also acts as a chemoattractant for myeloid-derived suppressor cells (MDSCs).^34,35 Since MDSCs contribute to the inhibitory milieu of the tumor micro environment (TME),^36,37 it is unclear how GM-CSF splays the balance between activation and inhibition in vivo.

Clinical studies have incorporated GM-CSF with the hope that it would be immune activating. Correlative studies in melanoma, breast and prostate cancer have not demonstrated a clear role for GM-CSF in antitumor immunity.^38-40 The E4697 phase III, double-blinded, placebo-controlled trial evaluated a peptide vaccine +/− GM-CSF (or placebo) in patients with completely resected stage IV or high-risk stage III melanoma did not show a survival benefit.⁴¹ Furthermore, a systematic review of 26 studies which evaluated the addition of GM-CSF to various treatment modalities including chemotherapy, surgery and peptide vaccines in patients with advanced melanoma did not show a significant improvement in outcomes with the addition of GM-CSF to peptide vaccines.⁴² The impact of GM-CSF's co-administration with the PROSTVAC vaccine in a completed Phase III study (NCT01322490) is under analysis. However, current evidence suggests GM-CSF is an inert companion for vaccines.

Vaccine plus IL-2

Interleukin-2 (IL-2) promotes differentiation of immature T-cells into both Tregs and effector T-cells.⁴³ Despite known activating effects on Tregs, high dose recombinant human IL-2 has produced durable remissions in select metastatic melanoma and metastatic renal cell carcinoma (mRCC) patients leading to FDA approval.^44-46 Studies of various vaccine platforms plus both low- and high-dose IL-2 have shown mixed results in recent years. The severe toxicity seen with higher doses has limited its use to select patients.⁴⁵

A phase I/II trial of DC vaccine plus low-dose IL-2 in patients with mRCC or breast cancer showed that the combination was well tolerated but there were no observed clinical responses.⁴⁷ A phase III trial randomized 185 patients with locally advanced stage III and stage IV cutaneous melanoma to gp100 peptide vaccine plus high-dose IL-2 vs high-dose IL-2 alone.⁴⁸ The combination group had better overall clinical response (16% vs. 6%, p = 0.03) and modest extension of PFS (2.2 months vs. 1.6 months, p = 0.008). Given the negative studies of low-dose IL-2 and high toxicities seen with high-dose IL-2, a future role for IL-2 in vaccine formulations does not appear likely as agents targeting downstream targets with less toxicity become available.

Vaccine plus IL-7

Interleukin-7 (IL-7) is important for differentiation of hematopoietic stem cells into lymphoid progenitor cells and development of CTL responses. Preclinical data show that PBMCs subjected to two-step culturing involving neoantigens exposed to GM-CSF followed by IL-7 produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific T-cells.⁴⁹ Other cytokine combinations were initially proliferogenic, but only IL-7 resulted in a sustained response.⁴⁹ An ongoing phase III trial is testing the combination of Sipuleucel-T plus subcutaneous IL-7 (CTY107), with the aim of augmenting proliferation of T-cell clones (NCT01881867).

Vaccine plus TGF-β modulation

Transforming growth factor β (TGF-β) is an important regulator of the cell cycle and is known to promote epithelial to mesenchymal transition (EMT).⁵⁰ Parts of the TGF-β signaling pathway are mutated in many malignancies, allowing invasion and metastasis, while TGF-β stimulation increases recruitment of MDSCs and Tregs.^50,51

A neoadjuvant clinical trial of an allogeneic pancreatic adenocarcinoma vaccine containing GM-CSF (GVAX) produced tertiary lymphoid aggregates (TLAs) within TME. Microarray studies of TLAs from patients who survived greater than 3 years showed suppression of multiple portions of the TGF- β pathway.⁵² Several small molecule TGF-β inhibitors are now under investigation and offer a means to dampen the immunosuppressive milieu within the TME. Galunisertinib has a safe toxicity profile based on two phase II trials in HCC and pancreatic cancer with no cardiac toxicity, which was a concern with first-generation TGF- β inhibitors.^53,54 To the best of our knowledge, no TGF-β inhibitors are currently being studied in combination with cancer vaccines. However, a novel bifunctional fusion protein called M7824 which consists of and anti-PD-L1 antibody and the extracellular domain of TGF-β receptor type two is currently being evaluated in a phase I trial in solid tumors. The receptor portion of the molecule essentially traps TGF-β and holds great promise as a companion to vaccine therapy (NCT02517398).

Vaccines plus radiotherapy

Rationale for combining radiotherapy (RT) with vaccines is multifold. Preclinical data have demonstrated that the combination of vaccines and RT is additive, with enhanced destruction of tumor cells via upregulation of MHC, Fas, ICAM-1 and TAAs, as well as by enhancing vaccine-mediated tumor lysis in mouse models.^55-57 Other studies have produced dramatic reduction in tumor burden in mice with combination RT plus vaccine, but not with either therapy alone, suggesting synergy.⁵⁸ T-cells specific for antigens not included in vaccine were also generated with combination, a phenomenon known as antigen cascade or antigen spreading. Moreover, non-lethal doses of radiation administered to tumor cell lines have also been shown to induce such phenotypes.^59,60 Lower doses of radiation can induce changes in tumor cells that make them more susceptible to T-cell killing through increased type I IFN secretion and increased expression of surface calreticulin.^29,61 The radiopharmaceutical, samarium-223, has been shown to have similar effects in vitro.⁶² Additionally, the abscopal effect, wherein non-irradiated lesions regress following radiation to a distant area, has also been reported in multiple malignancies,^63-66 including melanoma⁶⁷ and non-small cell lung cancer (NSCLC).⁶⁸

Since RT is a part of standard of care in many malignancies, many ongoing trials combining radiation and vaccine are designed to examine a vaccine's role in enhancing responses with RT as opposed to examining how RT may positively affect response to vaccines. Below is a brief summary of clinical experiences and ongoing trials.

Vaccines plus external beam radiotherapy

A phase I trial published in 2005 demonstrated increased PSA-specific T-cells in patients with locally invasive prostate cancer treated with RT plus a first generation fowlpox vaccine.³⁰ Patients were randomized to recombinant vaccinia (rV) -PSA, rV-B7.1 vaccine followed by monthly booster vaccines with recombinant fowlpox (rF)-PSA plus standard of care RT (19 patients) or RT alone (11 patients). Of the 17 patients in the combination arm who received all scheduled vaccinations, 13 had a ≥ 3-fold increase in PSA-specific T-cells. There was no such signal in the RT alone arm (p < 0.0005).

Several ongoing phase I studies are also evaluating the safety and efficacy of this approach. Self-adjuvanting mRNA cancer vaccine (RNActive®), called CV9202, targeting NY-ESO-1, MAGEC1, MAGEC2, 5 T4, survivin, and MUC1 is being tested in combination with RT for stage IV NSCLC (NCT01915524).⁶⁹ Preliminary data presented at the ASCO 2016 meeting demonstrated safety of CV9202 vaccine in combination with RT.⁷⁰ A personalized neoantigen vaccine for O⁶-methylguanin-DNA-methyltransferase (MGMT) unmethylated glioblastoma (GBM) in combination with RT is currently ongoing (NCT02287428). G207 is an oncolytic herpes simplex virus-1 engineered to contain mutations that enable it to selectively replicate within and kill cancer cells, but not normal cells.⁷¹ A phase I study indicated that G207 injected into recurrent high grade gliomas alone, or in combination with a single dose RT is well tolerated and active.^72,73 Another phase I study in pediatric patients with recurrent or progressive supratentorial tumors is currently recruiting subjects and will test G207 as monotherapy or combined with single dose RT (NCT02457845).

Another prostate cancer vaccine, aglatimagene besadenovec (ProstAtak®), is being evaluated in combination with RT in a phase III trial in patients with intermediate-high risk localized prostate cancer (NCT01436968). ProstAtak® is a cytotoxic immunotherapy derived from an adenovirus thymidine kinase (AdV-tk) vector that delivers herpes simplex virus into tumor cells when injected locally, creating a vaccine-like effect.⁷⁴ It utilizes valacyclovir as a prodrug. Patients receive three intra-prostate ProstAtak® treatments administered via transrectal ultrasound starting between 15 days and 8 weeks after beginning standard of care RT. Results are pending.

Vaccines plus radiopharmaceuticals

Samarium-153 EDTMP (¹⁵³Sm) is a radiopharmaceutical targeted to osteoblastic lesions. A phase II trial randomized 44 mCRPC patients previously treated with docetaxel to ¹⁵³Sm with or without PSA-TRICOM. The median PFS was 3.7 months for the combination vs 1.7 months for ¹⁵³Sm alone (HR 0.51; p = 0.041). Although the results were not statistically significant, there was a trend of decreased RDP and increased PFS.^75,76 A phase II study in mCRPC with bone metastases is currently recruiting patients who are randomized to Sipuleucel-T with or without radium-223 (the FDA approved radiopharmaceutical which demonstrated improvements in OS in mCRPC) (NCT02463799).

Vaccine plus checkpoint inhibitors

Vaccines plus anti-CTLA-4

CTLA-4 is expressed on T-cells and mediates inhibitory effects on CD4 helper T-cells during interactions with antigen presenting cells, representing an important mechanism of autoregulation.⁷⁷ CTLA-4 signaling can also have activating effects on Tregs.⁷⁸ CTLA-4 blockade with monoclonal antibodies is a potential strategy for converting vaccine-generated immune responses into clinically significant ones.

Ipilimumab and tremelimumab are anti-CTLA-4 antagonist monoclonal antibodies. Single agent use of ipilimumab has produced dramatic improvement in OS in advanced melanoma and is now FDA approved. However, ipilimumab has failed to achieve comparable clinical results in other solid tumors. For example, two phase III trials using ipilimumab in mCRPC failed to improve OS.^79,80 Both ipilimumab and tremelimumab are at various stages of clinical investigation alone or in combination with cancer vaccines.

In a phase I dose-escalation trial, a fixed dose of PROSTVAC was tested with escalating doses (1, 3, 5, and 10 mg/kg) of ipilimumab in mCRPC patients.⁸¹ There were no increases in immune-mediated AEs with combination. Fourteen of the 24 chemotherapy-naïve patients had a PSA decline with 6 patients having a PSA decrease > 50%. The median OS chemotherapy-naïve patients was 31.3 months, which was longer than historical controls of PROSTVAC alone.³⁸ There was a trend toward improved OS and the presence of certain immune cell subsets in peripheral blood.⁸² A trial testing tremelimumab in combination with several other agents, including a vaccine, is currently recruiting patients with mCRPC (NCT02616185).

Ipilimumab may also prove effective in a host of other malignancies and/or with other vaccine platforms, as illustrated by a study in 30 previously treated pancreatic adenocarcinoma randomized patients 1:1 to ipilimumab or ipilimumab plus GVAX.⁸³ No patients in the ipilimumab alone arm had a biochemical CA19–9 response, and two patients had stable disease (7 and 22 weeks). However, in the combination arm, 3 patients had prolonged stable disease (31, 71, and 81 weeks) and seven patients had a decline in CA-19–9. One year OS also favored combination (7 vs. 27%).⁸⁴ A phase III study showed that patients with advanced melanoma who received ipilimumab with or without the gp100 peptide vaccine (HLA-A*0201–restricted peptides derived from the melanosomal protein, glyco- protein 100) had OS of 10 months compared to patients who only received the gp100 vaccine (6.4 months).⁸⁵

As mentioned above, CTLA-4 mediates inhibitory effects on CD4 helper T-cells during interactions with antigen presenting cells.⁷⁷ For this reason, combining CTLA-4 blockade with antigen presenting cell administration i.e. DC vaccines is an exciting strategy. In a phase II trial, 39 pretreated advanced melanoma patients were given TriMixDC-MEL (autologous DC melanoma vaccine) intravenously and subcutaneously plus ipilimumab 10mg/kg every 3 weeks for four treatments. Patients who remained progression-free received maintenance therapy every 12 weeks.⁸⁶ The primary endpoint was met with a disease control rate of 51% at 6 months. Seven complete responses (CR) and 1 partial response (PR) were observed. The TriMixDC-MEL plus Ipilimumab ORR of 38% was better than monotherapy with ipilimumab in this population (10–15%) and was comparable to the ORR seen with anti-PD-1 monotherapy (ORR 25–43%) but not as high as seen in patients with dual anti-CTLA-4 plus anti-PD-1 therapy (ORR 57–61%).^87-90

Vaccines plus PD-1/PD-L1 inhibitors

Programmed cell death protein 1 (PD-1) is expressed on T-cells, as well as some B cells and NK cells, and binds to PD-L1 and PD-L2.⁷⁷ Antagonist antibodies that target the PD-1/PD-L1 axis have also achieved impressive and durable results in many solid tumors. Since 2015, multiple PD-1/PD-L1 inhibitors have received FDA approval for use in metastatic squamous NSCLC (nivolumab, pembrolizumab and atezolizumab), mRCC (nivolumab) unresectable/metastatic melanoma (pembrolizumab, nivolumab), locally advanced/metastatic urothelial carcinoma (atezolizumab, nivolumab, pembrolizuman, avelumab and durvalumab), recurrent/metastatic head and neck squamous cell carcinoma (pembrolizumab, nivolumab) metastatic merkel cell carcinoma (avelumab).⁹¹

Preclinical data shows that combination of an anti-PD-1 antibody and a multi-peptide vaccine (immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin) prolonged PFS in mice with breast tumors.⁹² There are several immune cell interactions that can be affected by PD-1/PD-L1 axis inhibition. Blockade of either of these targets can prevent PD-1/PD-L1 interaction-mediated inhibition of cytotoxicity at the effector cell:tumor cell synapse. Exerting a negative effect on each of these interactions with PD-1/PD-L1 blockade is promising as a means to enhance clinical activity of anti-tumor vaccines.⁹³

The gp100 peptide vaccine failed to enhance the clinical benefit produced by nivolumab in advanced melanoma patients.⁹⁴ However, nivolumab plus a multipeptide vaccine produced promising DFS data in the adjuvant setting for patients with high-risk resected melanoma. Median relapse-free survival (RFS) was 47.1 months with this combination which was highly favorable compared to historical median RFS (5 to 7.2 months) with other approaches.⁹⁵ Correlative studies were consistent with antigen-specific immune responses, and a trend towards lower levels of MDSC and Tregs was seen in non-relapsing patients. Tumor PD-L1 expression did not correlate with outcomes.

Preliminary clinical data combining a DNA vaccine encoding PAP in combination or in sequence with pembrolizumab were presented in November 2016 and showed promising results (NCT02499835).⁹⁶ Four out of six mCRPC patients treated with combination showed a decline in PSA, and imaging in 3 of 6 patients showed decreased tumor volume at 12 weeks.⁹⁶

Combination of vaccines and anti-PD-1 treatment are also underway in NSCLC. Viagenpumatucel-L is a cell based vaccine derived from a gp96-Ig secreting NSCLC tumor cell line selected for its ability to induce antigen specific T-cells.⁹⁷ A phase II study stratifying patients by high or low volume of TILs is currently enrolling patients and will test the vaccine in combination with nivolumab (NCT02439450). As seen with anti-CTLA-4 monoclonal antibodies, anti-PD-1 and anti-PD-L1 agents are also being tested in combination with different vaccine platforms for many malignancies (clinicaltrials.gov). Results are pending.

Vaccines plus small molecules

Tyrosine kinase inhibitors

Tyrosine kinase inhibitors (TKIs) are utilized in the treatment of several malignancies. VEGF-targeting TKIs (sunitinib, pazopanib, axitinib), multi-targeted TKI cabozantinib, and lenvatinib plus everolimus are FDA approved for treatment of mRCC. While these agents can improve outcomes, and produce objective responses, TKI's have not produced the durable CRs that have been observed in some mRCC patients treated with high-dose IL-2 therapy.^98-102

Growing preclinical and clinical data suggest that these agents often have “off target” effects on immune cells that enhance and/or damper the antitumor response.^103,104 For example sunitinib (inhibits KIT, PDGFR, KDR kinases, FLT3 kinase) decreases Tregs, decreases MDSCs, increases interferon-γ producing T-cells, and decreases IL-4 producing T-cells – all of which are important for an effective anti-tumor immune response.¹⁰⁵ Complicating the picture, sunitinib has also been found to inhibit activation of peripheral T-cells.¹⁰⁶ In a mouse model, sunitinib plus CEA-TRICOM vaccine decreased the angiogenesis in central areas of tumor whereas sunitinib alone only decreases angiogenesis in the periphery.¹⁰⁷ Furthermore, the combination of sunitinib or sorafenib plus CEA-TRICOM decreased tumor volume and intratumoral pressure. These effects on tumor vasculature appear to enhance migration of effector T-cells into the TME.¹⁰⁴

Another TKI, cabozantinib (inhibits c-MET, VEGFR2) also appears to have anti-tumor immune effects based on preclinical studies. It sensitizes murine tumor cells to immune-mediated killing, and when combined with CEA-TRICOM vaccine, reduces Treg and MDSC infiltration of the TME. As with sorafenib and sunitinib, cabozantinib plus CEA-TRICOM appears to normalize tumor vascularity favoring immune migration.¹⁰⁸ For these reasons, and their effectiveness as monotherapy, combining vaccines with standard of care TKIs is an attractive potential means to enhance their activity.

AGS-003 is a dendritic cell vaccine derived by co-electroporation of DCs with the patient's amplified tumor RNA and synthetic CD40L RNA. A phase II study treated 22 mRCC patients with AGS-003 plus sunitinib.¹⁰⁹ The vaccine was well tolerated. Out of 21 evaluable patients, 9/21 (43%) had a PR and 4/21 (19%) had SD. Median OS was 30.2 months, comparing favorably to historical data for mRCC patients treated with bevacizumab (22 months).¹¹⁰ A randomized phase III trial of AGS-003 plus sunitinib vs sunitinib alone is in progress (NCT01582672).

Vaccines with HDACi

Epigenetic silencing of genes involved in the immune-response, is one mode of immune escape utilized by tumor.¹¹¹ Histone deacetylase 1 inhibitors (HDACi) such as vorinostat and entinostat have been shown to sensitize tumor cells to antigen-specific T-cell mediated lysis and to increase the protein expression of antigen presenting machinery in breast cancer and prostate cancer cell lines.¹¹² While HDACi are promising, researchers have demonstrated enhanced cell migration and metastasis with use of HDACi in some human cancer cell lines and in mice.¹¹³ HDACi have little activity as single agents but have demonstrated efficacy when combined with cytotoxic and non-cytotoxic chemotherapy agents.¹¹⁴ Due to the immunomodulatory properties of HDACi there is growing interest using HDACi in combination with various drugs including checkpoint inhibitors and vaccines.

Vaccines plus endocrine therapy

In hormonally-driven tumors such as prostate cancer and breast cancer, the hormonal milieu is important in cancer development and progression. Breast cancer patients who received the aromatase inhibitor letrozole were found to have fewer Tregs within the TME.¹¹⁵ Furthermore, androgen deprivation in prostate cancer creates an immunostimulatory atmosphere, induces thymic regeneration and increases the number of effector T-cells.^116-118

The E9802 phase II trial tested the PROSTVAC vaccine followed by anti-androgen therapy in non-metastatic prostate cancer patients with biochemical recurrence (BCR). The combination was well tolerated. An increase in PSA doubling time between pre- and post-vaccine administration was observed (5.3 to 7.3 months).¹¹⁹ Notably, timing of vaccine treatment appears to be important. The survival data from a phase II trial suggested clinical benefit if vaccine was given prior to ADT. Survival analyses revealed a median OS advantage for the patients initially randomized to the vaccine arm who later received nilutamide compared to patients who received nilutamide first followed by vaccine (6.2 versus 3.7 years; p = 0.045).¹²⁰

Two phase II trials studying the androgen receptor antagonist, enzalutamide, with and without PROSTVAC in early and metastatic prostate cancer are ongoing. (NCT01867333, NCT01875250). The combination of vaccine with hormonal therapy in breast and prostate cancer is attractive since vaccines are minimally toxic and can easily be incorporated in standard of care regimens.

Vaccines plus chemotherapy

Many cytotoxic agents cause DNA damage or alter tumor phenotype, making the tumor more susceptible to CTL killing.^121,122 Chemotherapy agents such as docetaxel may have indirect effects on the immune system that improve their efficacy.¹²³ Chemotherapy dosing at maximum tolerated dosing (MTD) results in depletion of T-cells with both CD4+ and CD8+ cells effected but CD8+ cells recover more quickly.¹²⁴ In addition, NK cells are also impaired. Standard of care dosing for most chemotherapies is often much lower than the MTD and allows for a residual immune response.¹²³ Several chemotherapeutic agents, including gemcitabine, taxanes, topoisomerase inhibitors, platinum compounds, and 5-FU have been shown to produce immunomodulatory effects.^{121,123,125,126} These effects are discussed in more detail below.

Vaccine plus low dose cyclophosphamide (CTX)

Metronomic (low) dosing of CTX has been shown to inhibit Tregs,¹²⁷ enhance DC maturation, and promote memory T-cell responses, making it a candidate for enhancing cancer immunotherapies.^122,128 A single-arm feasibility study gave HER2+ metastatic breast cancer patients allogeneic HER2+ GM-CSF-secreting whole-cell breast cancer vaccine one day after receiving CTX 300 mg/m² and trastuzumab 2mg/kg (NCT00399529). All patients were HER2+ and 13 of 20 patients were hormone receptor positive. The clinical benefit rate (CBR = CR+ PR+SD) at 6 months was 55% (p = 0.013) and at 12 months was 40%.¹²⁹

Results from an international phase II/III study in metastatic breast cancer patients testing CTX plus OPT-822, a vaccine targeted to a glycolipid overexpressed in breast cancer, or placebo were presented at ASCO 2016 (NCT01516307).¹³⁰ Similar to the above study, the vaccination plus CTX arm did not improve PFS or OS. However, PFS and OS were higher in patients who developed an immune response to the vaccination.¹³⁰ There is currently an ongoing phase II study is comparing viagenpumatucel-L plus CTX to CTX alone in patients with advanced NSCLC who have failed multiple prior therapies (NCT02117024).

Vaccines plus gemcitabine

Preclinical studies have shown that gemcitabine can increase antigen cross-presentation, decrease MDSC and Tregs, and increase immune-supportive M1 macrophages, circulating CD4+ and CD8+T cells^131,132 These effects were observed in human in a phase I/II trial in ovarian cancer patients treated with gemcitabine, Pegintron and p53 synthetic long peptide (SLP) vaccine.¹³³ However, effects on outcome in patients treated with vaccine plus gemcitabine have been mixed. Results from a phase II study of algenpantucel-L plus gemcitabine and 5-fluorouracil-based standard adjuvant chemoradiotherapy for resected pancreatic cancer demonstrated 12-month PFS of 62% and 12-month OS of 86%.¹³⁴ While these compared favorably to historical data,¹³⁵ a phase III study randomized patients 1:1:1 to receive chemotherapy alone, chemotherapy followed by a telomerase vaccine (GV1001), or concurrent chemotherapy and vaccine in metastatic pancreatic cancer did not meet its primary OS endpoint.^136,137

Vaccines plus docetaxel

Combination of vaccine plus docetaxel has been shown to antigen specific CD8 T-cells and decrease tumor burden in murine models.¹³⁸ Additionally, other preclinical data suggest docetaxel can make tumor cells more susceptible to CD8 T-cell-mediated cytotoxicity via enhanced calreticulin expression.³¹

A phase II trial evaluated docetaxel alone vs modified vaccinia Ankara vaccine (TroVax; targeted tumor antigen 5T4) followed by docetaxel in patients with mCRPC. Although the study was closed prematurely due to accrual issues with only 25 patients enrolled, a superior median PFS of 9.67 months was observed in the TroVax + docetaxel compared to 5.1 months in the docetaxel alone arm (p = 0.097).¹³⁹

A phase II trial evaluated docetaxel alone verse docetaxel combined with PANVAC (contains MUC-1, CEA and co-stimulatory molecules B7.1, ICAM-1, LFA-3) in metastatic breast cancer patients.⁴⁰ Forty-eight patients were enrolled; 23 were randomized to docetaxel alone and 25 were randomized to combination arm. A significant increase in median PFS was observed in the PANVAC plus docetaxel vs. the docetaxel only arm (7.9 months vs 3.9 months; p  =  0.09).⁴⁰ Secondary analyses demonstrated no correlation between generation of the T-cell specific immune response and time to progression.⁴⁰ Patients who received only docetaxel also developed T-cell responses to the TAA supporting the hypothesized immunomodulatory properties of docetaxel.

Several ongoing studies are currently evaluating combination of docetaxel and different vaccines in prostate cancer: PROSTVAC plus docetaxel in castration sensitive prostate cancer (phase II; NCT02649855) and docetaxel plus DCVAC/PCa in metastatic castration resistant disease (phase III; NCT02111577).

Vaccine plus irinotecan

Irinotecan blocks DNA repair and stimulates a complex immune response including activation of tumor-suppressor proteins and induction of tumor immunosurveillance by NK cells and activated CD8+ T-cells.¹²¹

A phase II multicenter study tested G17DT (a vaccine consisting of the N-terminus of gastrin 17, a growth factor, conjugated to diptheroid toxin that elicits anti-gastrin 17 antibodies) plus irinotecan in metastatic CRC patients who were progressing on irinotecan. Of the 161 patients, PR was observed in 3%, SD in 32% and PD in 65% of treated patients. Aside from increased injection site reactions seen in 52% of patients, the side effect profile was similar to irinotecan alone. Sixty-two percent of patients had measurable anti-gastrin 17 antibodies, which was associated with a survival benefit (9.0 vs. 5.6 months; p < 0.001).¹⁴⁰

Vaccine plus platinum-based chemotherapy

Carboplatin-paclitaxel combination also has immunomodulatory anti-tumor effects in preclinical studies.¹²¹ Recently published work demonstrated that carboplatin-paclitaxel plus HPV16 peptide vaccine increased survival in murine tumor models, correlating with decreased number of myeloid cells in tumor and peripheral blood.¹⁴¹ Additionally, carboplatin-paclitaxel increased ex vivo T-cell activity to recall antigens.

A phase 2b/3 trial in the first line setting for stage IV NSCLC showed some benefit with TG4010 (modified Ankara vaccine expressing MUC-1 and IL-2) added to platinum-based chemotherapy.¹⁴² Patients were randomized to TG4010 plus chemotherapy (n = 111) or placebo plus chemotherapy (n = 111). The combination group had a longer median PFS (5.9 vs 5.1 months; p = 0.019) and more confirmed responses (40% vs 29%, respectively). Interestingly, there were delayed responses and more durable responses observed in the TG4010 group (median duration was 30.1 weeks in responders who received TG4010 and 18.7 weeks in placebo group responders).¹⁴³

Expert opinion

As detailed above, there are many vaccine platforms and target antigens to be tested in different malignancies and different clinical settings. Despite intensive study during the past 20 years, it was not vaccines, but immune checkpoint inhibitors that first revolutionized cancer therapy by altering the treatment landscape in many cancers including melanoma, non-small cell lung cancer, and urothelial carcinoma.^85,144,145 The three approved cancer vaccines have produced only modest improvements in OS (sipuleucel-T, T-VEC), DFS (TheraCys) and DRR (T-VEC) in patients with early stage or a limited disease burden. If there is to be an therapeutic role for vaccines especially in more diverse clinical settings and in patients with advanced or metastatic cancers, it will likely be as part of combination therapy.²⁶ While marveling at the successes of checkpoint inhibitors, it is important to bear in mind that these therapies to date have not worked in most malignancies or even provide benefit for the majority of patients with malignancies in which these agents are known to be active. The biology of resistance to these therapies is far from being understood, but one possible explanation is a lack of tumor-specific T-cells, leaving the checkpoint inhibitors without any effector cells to ‘unleash.’ For the following reasons, vaccines may be able to correct this deficit.

We know that various vaccine platforms are capable of generating tumor-specific T-cells, and in some cases, have been shown to increase tumor infiltrating T-cells.¹⁴⁶ Findings from early phase studies support the hypothesis that vaccines plus checkpoint inhibitors can create a situation for success where there may have been none with monotherapy. For example, in mCRPC, ipilimumab plus PROSTVAC appears to have a survival benefit compared to historical controls of ipilimumab alone.^38,81 Ipilimumab plus GVAX produced periods of SD for 3 pancreatic adenocarcinoma patients (31, 71, and 81 weeks), compared to SD for 2 patients (7 and 22 weeks) with ipilimumab alone.⁸³ The phase II TriMixDC-MEL plus ipilimumab in melanoma also produced comparable or superior response rates compared to phase II data of some checkpoint inhibitors.^86-90 Early phase data testing nivolumab plus vaccine in the adjuvant setting for high risk melanoma compare favorably to historical relapse free survival data.⁹⁵ In making the argument for the promise of vaccine plus checkpoint inhibitors, we have intentionally included examples from different malignancies. This highlights the need to consider available preclinical data, relevant to an individual tumor type's biology, when selecting agents for combination with vaccine.

Furthermore, vaccines will be enhanced as antigen selection is refined. The potential influence of neoantigen expression on response to checkpoint inhibitors¹⁴⁷ has been noted, however the exact role(s), mechanism(s), and utility as target antigens are yet unclear. Utilizing high throughput whole exome and transcriptome sequencing techniques, as described by Yadav et al.²⁵ offers a means to identify highly immunogenic antigens for incorporation into any number of the vaccine platforms described above. As suggested by others, personalized tumor vaccines, based on synthesized neoantigens derived from whole tumor exome sequencing or use of autologous tumor for antigen sourcing, should be a prioritized strategy.²⁶ The potential power of combining such ‘smart target’ vaccines with checkpoint inhibitors remains untested but holds great promise.

Conclusion

Experience suggests that the therapeutic cancer vaccines currently in clinical development are unlikely to dramatically impact cancer outcomes as single agents. Several combination approaches including vaccine plus cytokines, checkpoint inhibitors, small molecule inhibitors, radiotherapy and chemotherapy have been tested. Based on these findings, it appears that combining therapeutic vaccines with immune checkpoint inhibitors holds the greatest potential for improving clinical outcomes. Addition of vaccines to various cancer treatment modalities can augment immune responses with minimal additional toxicity. Additionally, optimizing target antigen selection via novel high throughput sequencing is a developing technique that has generated excitement. Traditional study endpoints and assessment criteria may not be appropriate given the different mechanisms of action and response patterns seen with immunotherapy agents. As we learn more about the mechanisms of immune evasion, various therapies will be combined and perhaps specific sequencing of this multi-modality approach will prove to be superior for achieving durable responses. A one-size-fits-all approach is not effective and our goal should be to better understand the biology and to develop predictive biomarkers. Cancer vaccines are here to stay, as evidenced by the many ongoing clinical trials evaluating immunotherapy combinations.

Disclosure of potential conflicts of interest

No potential conflict of interest was reported by the authors.